Group of markers capable of reflecting post-PCI pro-inflammatory phase prolongation of ACS patient and application thereof

Abstract

The invention belongs to the technical field of biomarkers, and particularly relates to a group of markers capable of reflecting post-PCI proinflammatory phase extension of ACS patients and application of the markers. The marker provided by the invention comprises butyryl carnitine, valeryl carnitine, propionyl carnitine, hydroxyisovaleryl carnitine and linoleyl carnitine, the content of the marker in plasma can represent pro-inflammatory phase extension and anti-inflammatory repair phase postponing phenomena related to pro-inflammatory Mo high level in plasma after PCI of an ACS patient, and the myocardial ischemia injury degree of the ACS patient can be reflected in time.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarkers, and specifically discloses a group of markers that can reflect the extension of the pro-inflammatory phase of ACS patients after PCI and their application. Background Art

[0002] Acute coronary syndrome (ACS) is a group of clinical syndromes caused by acute myocardial ischemia. Although great progress has been made in the diagnosis and treatment of ACS, the proportion of ventricular remodeling in ACS patients who have successfully undergone percutaneous coronary intervention (PCI) is very high, and the proportion of ventricular remodeling in ACS patients in my country is as high as 30% to 60%. Ventricular remodeling after ACS is one of the main causes of heart failure (HF).

[0003] The regulation of adaptive remodeling and adverse remodeling is the key to preventing and treating ventricular remodeling after ACS. The innate immune response is an important regulator between adaptive remodeling and adverse remodeling, including three key stages: proinflammatory phase, anti-inflammatory repair phase and mature phase, among which the balance between proinflammatory phase and anti-inflammatory repair phase is an important link in regulating adaptive remodeling and adverse remodeling. Prolongation of the proinflammatory phase occurs when there is an imbalance in the conversion between the proinflammatory phase and the anti-inflammatory repair phase. Due to the overexpression of immune cells and excessive degradation of the extracellular matrix, the damaged area will be expanded, the anti-inflammatory repair phase will be delayed, the formation of scar tissue will be hindered, and the adverse left ventricular remodeling will be aggravated, leading to ventricular remodeling after ACS.

[0004] Due to the complex interaction between the proinflammatory phase and the anti-inflammatory repair phase, although many drugs that inhibit the proinflammatory phase and reduce the inflammatory immune response have been tested in ACS patients, the results are unsatisfactory. Currently, drugs that specifically target the proinflammatory phase have adverse reactions such as increased fatal infections and even increased all-cause and non-cardiovascular mortality. Therefore, markers that can reflect the proinflammatory phase prolongation will provide a basis for judging ventricular remodeling and HF after ACS.

[0005] CK and cTnT are markers that reflect the degree of myocardial necrosis, but they cannot warn of myocardial ischemic injury in advance. In addition, the half-life of CK-MB is relatively short. For some AMI patients without obvious clinical manifestations, the best time for diagnosis may be missed, treatment may be delayed, and prognosis may be affected. cTnT is heart organ specific, not AMI specific. In the absence of myocardial necrosis, the increase of cTnT may be multifactorial, including myocardial ischemia, increased cardiac wall tension and ventricular strain, direct myocyte trauma, excessive catecholamines, and impaired renal clearance. In cardiovascular diseases, CRP can be used as an inflammation-related biomarker to reflect myocardial damage caused by myocardial ischemia and hypoxia, but there are obvious individual differences. For example, body mass index, metabolic syndrome, diabetes, hypertension, use of oral contraceptives, physical exercise, excessive drinking, periodontal disease, smoking, etc. can all lead to significant baseline changes, and the CRP levels vary greatly between different races and genders, so it does not have specificity and high sensitivity for diagnosis. Summary of the invention

[0006] In view of the above technical problems, the present invention provides a group of markers that can reflect the prolongation of the pro-inflammatory phase after PCI in ACS patients and their applications. The markers provided by the present invention can represent the phenomenon of prolongation of the pro-inflammatory phase and delay of the anti-inflammatory repair phase associated with high levels of pro-inflammatory Mo in the plasma of ACS patients after PCI, thereby being able to timely reflect the degree of myocardial ischemic injury in ACS patients, and have strong specificity and high sensitivity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a group of markers that can reflect the extension of the pro-inflammatory phase after PCI in ACS patients, including butyrylcarnitine, valerylcarnitine, propionylcarnitine, hydroxyisovalerylcarnitine and linoleoylcarnitine.

[0009] The present invention has found through research that the above-mentioned acylcarnitine is a plasma differential metabolite that can represent two time nodes of pro-inflammatory period extension and anti-inflammatory repair period delay associated with high level of pro-inflammatory Mo after PCI in ACS patients. Propionylcarnitine, butyrylcarnitine, valerylcarnitine and hydroxyisovalerylcarnitine increase when the level of pro-inflammatory Mo in peripheral blood of ACS patients is significantly high, and have a large positive correlation with the occurrence of cardiovascular events after ACS. Linoleoylcarnitine decreases when the level of pro-inflammatory Mo in peripheral blood of ACS patients is significantly high, and has a negative correlation with the occurrence of cardiovascular events after ACS.

[0010] The changes in metabolic pathways from significantly high levels of pro-inflammatory Mo to delayed anti-inflammatory repair after PCI in ACS patients mainly include glucose homeostasis, fatty acid β-oxidation, lipid metabolism, ATP energy metabolism, etc. The above five acylcarnitines are all products of fatty acid β-oxidation and are related to fatty acid metabolism disorders in energy metabolism disorders. The animal model verified that the change trends of the above five acylcarnitines in plasma and heart tissue of the C57BL / 6J mouse model of left anterior descending coronary artery ligation 45 minutes after reperfusion were consistent with the clinical research results.

[0011] Therefore, the above five acylcarnitines can be used as markers for the prolonged proinflammatory phase after PCI in ACS patients, and their levels in plasma can reflect the degree of myocardial ischemia in ACS patients, thus being able to be used to determine whether ventricular remodeling occurs after PCI in ACS patients, facilitating clinical intervention. Moreover, the above five acylcarnitines reflect the degree of myocardial injury earlier than CK-MB and cTnT.

[0012] Preferably, the marker also includes at least one of N-acetyl-L-methionine, lysophosphatidylethanolamine (18:2), lysophosphatidylethanolamine (20:2), lysophosphatidylethanolamine (22:6), L-isoleucine, hypoxanthine, N-docosahexaenoic acid GABA, tetrahydroaldosterone-3-glucuronic acid, lysophosphatidylcholine (18:2), glycocholic acid and creatinine. Among them, N-acetyl-L-methionine, lysophosphatidylethanolamine (18:2), lysophosphatidylethanolamine (20:2), lysophosphatidylethanolamine (22:6), L-isoleucine and hypoxanthine increase when the level of proinflammatory Mo in the peripheral blood of ACS patients is significantly high, while N-docosahexaenoic acid GABA, tetrahydroaldosterone-3-glucuronic acid, lysophosphatidylcholine (18:2), glycocholic acid and creatinine decrease when the level of proinflammatory Mo in the peripheral blood of ACS patients is significantly high. These markers can be used together with the above five markers to reflect the high level of proinflammatory Mo in the peripheral blood, and thus reflect the degree of myocardial ischemic injury in ACS patients.

[0013] The second aspect of the present invention provides the use of the above markers in the preparation of a product reflecting the degree of myocardial injury in ACS patients.

[0014] The third aspect of the present invention provides the use of a reagent for detecting the above-mentioned marker in the preparation of a reagent or a kit reflecting the degree of myocardial injury in ACS patients.

[0015] The fourth aspect of the present invention also provides a method for detecting the above markers in plasma, which specifically comprises the following steps:

[0016] S1. Add internal standard working solution and glacial acetonitrile to the plasma to be tested, mix and incubate at -15 to -20°C for 1 to 1.5 h, centrifuge at 12000 to 15000 rpm for 20 to 30 min at 3 to 5°C, take the supernatant, dry and reconstitute with methanol-water solution, centrifuge at 12000 to 15000 rpm for 20 to 25 min at 3 to 5°C, and remove the supernatant as the test solution; the internal standard is deuterated cholic acid-[d 4 ];

[0017] S2. Detecting the solution to be tested by liquid chromatography-mass spectrometry technology, and determining the degree of myocardial damage according to the change in the content of the marker.

[0018] In S2, if the levels of propionylcarnitine, butyrylcarnitine, valerylcarnitine and hydroxyisovalerylcarnitine in the plasma of patients with acute coronary syndrome after PCI are significantly increased, and the level of linoleoylcarnitine is significantly decreased, it is judged that the proinflammatory phase is prolonged and ventricular remodeling may occur.

[0019] Preferably, the LC-MS technique uses UHPLC / Q-Orbitrap-MS.

[0020] Preferably, the chromatographic conditions of the UHPLC / Q-Orbitrap-MS include: the chromatographic column is HSS T3, the specifications are 2.1×100 mm, 1.8 μm, the mobile phase A is 0.1% v / v formic acid aqueous solution, the mobile phase B is acetonitrile, and the elution gradient is: 0-7 min, 5%-100% B; 7-10 min, 100% B; 10-10.5 min, 100%-5% B; 10.5-13 min, 5% B; the column temperature is 35° C., and the flow rate is 0.3 mL / min.

[0021] Preferably, the mass spectrometry analysis of the UHPLC / Q-Orbitrap-MS adopts a Q-ExactiveTM hybrid Q-Orbitrap mass spectrometer system.

[0022] Further preferably, the HESI source parameters for mass spectrometry analysis are: spray voltage -3.5 kV / +3.5 kV, sheath gas 35 L / h, auxiliary gas 10 L / h, purge gas 0 L / h, capillary temperature 320°C, and auxiliary gas heating temperature 350°C.

[0023] Further preferably, the mass spectrometry analysis adopts Full MS / dd-MS 2 Scanning method, MS 1 The full scan range is m / z 60-900, and the resolution is 70000; MS 2 The mass spectrometer was scanned over a dynamic mass range with a resolution of 17,500.

[0024] The beneficial effect of the present invention is that after ACS patients undergo PCI, there is still a high level of pro-inflammatory Mo, which prolongs the pro-inflammatory period, resulting in a delay in the anti-inflammatory repair period, affecting myocardial repair. The markers provided by the present invention, propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleylcarnitine, are related to fatty acid metabolism disorders in energy metabolism disorders, and can be used as markers for the pro-inflammatory period extension after PCI in ACS patients. By detecting the above markers in the plasma of ACS patients after PCI, it is possible to judge whether ventricular remodeling may occur in ACS patients after PCI by the marker content, so as to facilitate clinical intervention; and the content of the above markers can release risk prediction signals in the early stage of myocardial ischemic injury, detect and intervene as early as possible, and reduce the cost of clinical prevention and treatment of ACS and ventricular remodeling after ACS. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the proportion of Mo subgroups at different time points within 1 week before and after PCI in AMI patients in Example 1 of the present invention; A pro-inflammatory Mo (CD14 ++ CD16 - ), B is intermediate Mo (CD14 ++ CD16 + ), C is repaired Mo (CD14 + CD16 ++ ); "*" indicates P<0.05, "**" indicates P<0.01;

[0026] Figure 2 It is the result of multivariate statistical analysis of plasma metabolism changes when peripheral blood Mo subgroups change at different time points 1 week before and after PCI in AMI patients in Example 1 of the present invention;

[0027] Figure 3 is the result of multivariate statistical analysis of plasma metabolomics of AMI patients 7 days after PCI and healthy volunteers in Example 1 of the present invention;

[0028] Figure 4 is the volcano plot of differential metabolites in AMI patients Post-PCI 3d vs Post-PCI 7d in Example 1 of the present invention;

[0029] Figure 5 It is the metabolic pathway related to the differential metabolites of AMI patients Post-PCI 3d vs Post-PCI 7d in Example 1 of the present invention;

[0030] Figure 6It is the dynamic metabolic changes of the five acylcarnitines in the process of changes in the Mo subgroup of AMI patients at different time points before and after PCI in Example 1 of the present invention; "&" indicates a comparison with the Con group, P < 0.05, "*" indicates a comparison with Post-PCI 1d, P < 0.05, and "#" indicates a comparison with Post-PCI 3d, P < 0.05;

[0031] Figure 7 is the result of multivariate statistical analysis of plasma metabolic changes when peripheral blood Mo subpopulations change at different time points 1 week before and after PCI in UA patients in Example 1 of the present invention;

[0032] Figure 8 The results of multivariate statistical analysis of plasma metabolomics in UA patients 7 days after PCI and healthy volunteers in Example 1 of the present invention are

[0033] Fig. 9 The metabolic changes of the five acylcarnitines in Example 1 of the present invention during the changes in Mo subgroups at different time points before and after PCI in AMI and UA patients are

[0034] Fig.10 are ultrasound images of mice at different time points within 1 week before and after reperfusion in Example 2 of the present invention;

[0035] Fig.11 These are the ultrasound results of mice at different time points within 1 week before and after reperfusion in Example 2 of the present invention: compared with the Sham group, "*" indicates P < 0.05, and "**" indicates P < 0.01;

[0036] Fig.12 is the change of each marker in Example 2 of the present invention in mouse plasma at different time points;

[0037] Fig.13 This is a mass spectrum comparison of linoleylcarnitine and a standard product in the heart tissue of mice 3 days after ischemia-reperfusion in Example 2 of the present invention; A is the sample, and B is the standard product of linoleylcarnitine;

[0038] Fig.14 This is a secondary fragmentation diagram comparing linoleylcarnitine and a standard product in the heart tissue of mice 3 days after ischemia-reperfusion in Example 2 of the present invention; A is the sample, and B is the linoleylcarnitine standard product;

[0039] Fig.15 This is the relationship between the accumulation of linoleylcarnitine in cardiac tissue and the cardiac function of mice 3 days after ischemia-reperfusion in Example 2 of the present invention; "*" indicates that compared with the Sham group, P<0.05. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0041] ACS patients still have significantly high levels of proinflammatory Mo after PCI, which prolongs the proinflammatory phase and affects the repair phase. Myocardial necrosis is irreversible, but there is a reversible repair space between myocardial ischemia and necrosis. Clinically, markers that can reflect myocardial damage caused by myocardial ischemia and hypoxia are needed.

[0042] Monocytes (Mo) play an important role in both the proinflammatory and anti-inflammatory repair phases of cardiac remodeling after ACS. High levels of proinflammatory Mo in the peripheral blood of ACS patients are associated with prolonged proinflammatory phase and impaired left ventricular function recovery. Regulatory treatment of Mo and macrophage (Ma) polarization can provide a new therapeutic strategy for reducing myocardial infarction size and preventing ventricular remodeling after ACS. Targeting proinflammatory Mo or M1-type Ma to inhibit the proinflammatory phase after ACS has a cardioprotective effect. Clinical studies have shown that high levels of proinflammatory Mo in the peripheral blood of ACS patients are associated with prolonged proinflammatory phase and impaired left ventricular function recovery. Therefore, the discovery of substances that can reflect high levels of proinflammatory Mo is very important for studying the inhibition of prolonged proinflammatory phase after ACS and the prevention and treatment of ventricular remodeling after ACS.

[0043] Changes in cell phenotype and function are accompanied by dramatic changes in cell metabolism. Mo has metabolic adaptability, and its metabolic characteristics depend on the surrounding microenvironment. Therefore, its metabolism will change under different conditions, and some metabolic changes can also affect the function of immune cells. The present invention is based on flow cytometry detection technology and UHPLC / Q-Orbitrap-MS technology. Through research, it is found that the contents of 10 differential metabolites such as propionylcarnitine, butyrylcarnitine, and valerylcarnitine are significantly increased when the level of proinflammatory Mo in the peripheral blood of ACS patients is high, and significantly decreased at the time point representing the delay of the anti-inflammatory repair period; 6 differential metabolites such as linoleoylcarnitine, N-docosahexaenoic acid GABA, and lysophosphatidylcholine (18:2) are low in the level of proinflammatory Mo in the peripheral blood of ACS patients, and significantly increased at the time point representing the delay of the anti-inflammatory repair period. The changes in metabolic pathways from the significant high level of proinflammatory Mo to the delay of the anti-inflammatory repair period in ACS patients after PCI mainly include glucose homeostasis, fatty acid β-oxidation, lipid metabolism, ATP energy metabolism, etc. Among the above 16 differential metabolites, 5 acylcarnitines are products of fatty acid β-oxidation, namely propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleylcarnitine. After a comprehensive analysis of the dynamic metabolic changes in the Mo subgroups at different time points, it was found that propionylcarnitine, butyrylcarnitine, valerylcarnitine and hydroxyisovalerylcarnitine showed a change trend consistent with the proinflammatory Mo at different time points, while the change trend of linoleylcarnitine was opposite to the high level trend of the proinflammatory Mo. Therefore, propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleylcarnitine in the plasma of ACS patients after PCI can be used as markers of prolonged proinflammatory phase, and their contents can reflect the degree of myocardial ischemic injury in ACS patients.

[0044] Based on the above new research results, the embodiments of the present invention provide a group of markers that can reflect the extension of the pro-inflammatory phase after PCI in ACS patients.

[0045] The embodiment of the present invention also provides the use of the above markers in preparing a product reflecting the degree of myocardial injury in ACS patients.

[0046] The embodiment of the present invention also provides the use of a reagent for detecting the above-mentioned marker in preparing a reagent or a kit for reflecting the degree of myocardial injury in ACS patients.

[0047] The present invention also provides a method for detecting the above markers in plasma.

[0048] The scheme of the present invention is described below through specific embodiments.

[0049] The main analytical instruments used in the following examples are: flow cytometer, BD FACSVerse TM(BD Biosciences, USA), Ultimate 3000 ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA), high-resolution mass spectrometer Q-Exactive-Orbitrap MS (Thermo Fisher Scientific, Germany).

[0050] The main reagents used in the following examples: PerCP / Cyanine5.5anti-human CD45, APC / Cyanine7anti-human CD14, PE anti-human CD16, Human TruStain FcX TM ,PerCP / Cyanine5.5Mouse IgG1(κisotype Ctrl), APC / Cyanine7Mouse IgG1(κisotype Ctrl), PEMouse IgG1(κisotype Ctrl) were purchased from BioLegend, USA; sheath fluid for flow cytometry analysis, BD FACS cleaning solution, BD Extended Flow Cell Clean Solution, Ms Ig Kpa Comp Bead Set were purchased from BD Biosciences, USA; red blood cell lysis buffer, 4% tissue cell fixative were purchased from Beijing Solebow Technology Co., Ltd.; HPLC grade methanol, acetonitrile, and formic acid were purchased from Fisher, USA; propionylcarnitine standard was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine, and linoleylcarnitine standards were purchased from Sigma-Aldrich, Merck, USA; deuterated cholic acid-[d 4 ] was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0051] Unless otherwise specified, other raw materials, reagents, drugs or instruments used in the following examples are conventional commercial products obtained from commercial channels. The methods used in the following examples are conventional methods in the art unless otherwise specified.

[0052] Example 1

[0053] This embodiment provides markers that can reflect high levels of pro-inflammatory Mo and their use in reflecting high levels of pro-inflammatory Mo, and provides methods for detecting the above markers in plasma.

[0054] 1. Experimental methods

[0055] 1.1 Research subjects

[0056] 1.1.1 Source of research subjects

[0057] From May to December 2022, 223 ACS patients and 53 healthy volunteers were enrolled in the Emergency Department, Critical Care Unit of Coronary Heart Disease, and Second Ward of Cardiology Department of Yuhai District People's Hospital, Yuhai City (Tianjin, China). This trial was approved by the Management Committee of Yuhai District People's Hospital of Yuhai City (Approval No.: JHYYLL-2022-0121), and informed consent was obtained from the patients.

[0058] 1.1.2 Research Subject Selection Criteria

[0059] 1.1.2.1 Diagnostic criteria

[0060] AMI: ① Symptoms of acute myocardial ischemia such as chest pain occur within 24 hours before admission and last for more than 30 minutes, and sublingual nitroglycerin has no relief effect; ② New ischemic electrocardiogram changes; ③ Increased serum creatine kinase (CK) and cardiac troponin T (cTnT) levels.

[0061] UA: ① 48 days before admission

[0062] ② Symptoms of ischemic chest pain at rest within h; ② The electrocardiogram shows transient ST-T segment depression and / or T wave inversion, but there is no evidence of myocardial necrosis according to enzyme standards.

[0063] 1.1.2.2 Inclusion criteria

[0064] AMI group and UA group: ① met the diagnostic criteria of AMI and UA; ② AMI and UA patients received coronary angiography and PCI after admission; ③ received optimal drug treatment; ④ had good compliance with the study and signed written informed consent.

[0065] CON group: ① healthy patients, aged 18-80 years old; ② informed consent.

[0066] 1.1.2.3 Exclusion criteria

[0067] ① Allergy to iodine; ② Severe liver and kidney dysfunction; ③ Patients with concurrent infectious diseases, mental illness, impaired consciousness, active infection, severe organic lesions, dysfunction of important organs, autoimmune diseases, coagulation disorders, and malignant tumors; ④ Pregnant and lactating women; ⑤ People <18 years old or >80 years old.

[0068] 1.1.2.4 Elimination criteria

[0069] ① The clinical data were lost after enrollment; ② The patients had low cooperation or compliance with the study.

[0070] 1.1.3 Grouping of research subjects

[0071] According to the inclusion and exclusion criteria, the included patients were finally divided into three groups: AMI group (n = 170 cases), UA group (n = 53 cases) and Con group (n = 53 cases).

[0072] 1.2 Research Methods

[0073] 1.2.1 Clinical data collection

[0074] The gender, age, complications, other cardiovascular risk factors and medication status of the included patients, as well as the blood routine, liver and kidney function, blood lipids, blood sugar, electrocardiogram, cardiac ultrasound, etc. of all eligible patients were recorded.

[0075] The clinical characteristics of each group are shown in Table 1.

[0076] Table 1 Clinical data related indicators [x±s, M (Q1, Q3), n (%)]

[0077]

[0078]

[0079] Note: “*” indicates the comparison between AMI group and UA group, P<0.05.

[0080] 1.2.2 Peripheral blood sample collection

[0081] 2 mL of EDTA anticoagulated blood was collected from healthy volunteers on the day of physical examination and from ACS patients before and 1 d, 3 d, and 7 d after PCI, and transported to the laboratory in an ice box. 200 μL of whole blood was used for flow cytometry (tested within 24 h), and the remaining blood samples were centrifuged at 4°C, 3000 rpm, for 5 min. The supernatant was taken and transferred to 1.5 mL EP tubes (100 μL per tube) and stored in a -80°C refrigerator.

[0082] 1.2.3 Detection of surface molecule expression of peripheral blood monocytes by flow cytometry

[0083] The specific steps are as follows:

[0084] ① Take 200 μL of EDTA anticoagulated blood and add it to a 15 mL centrifuge tube and mix gently;

[0085] ② Before incubation with fluorescently labeled antibodies, add 10 μL of FcR blocking antibody to the centrifuge tube, vortex to mix, and incubate on ice in the dark for 20 minutes;

[0086] ④ Add 3 mL of D-hanks, mix well, and centrifuge (1400 rpm, 5 min, 4°C);

[0087] ⑤ Aspirate and discard the supernatant, add fluorescently labeled monoclonal antibodies (CD45-PerCP / Cyanine5.5, CD14-APC / Cyanine7, CD16-PE, 1 Test each) or isotype control antibodies (PerCP / Cyanine5.5, APC / Cyanine7, PE-Isotype control, 1 Test each), vortex to mix, and incubate on ice in the dark for 30 minutes;

[0088] ⑥ Add 3 mL of D-hanks, mix well, and centrifuge (1400 rpm, 5 min, 4°C);

[0089] ⑦ Aspirate the supernatant, add 2 mL of red blood cell lysis buffer, vortex to mix, and incubate at room temperature in the dark for 15-18 minutes. When the cell suspension changes from turbid to transparent, add 8 mL of D-hanks, mix, and centrifuge (1400 rpm, 5 min, 4°C);

[0090] ⑧Discard the supernatant and add 300 μL D-hanks to resuspend the sample;

[0091] ⑨ Before loading, filter the sample through a 70 μm filter into a flow tube and detect the expression of each molecule by flow cytometry;

[0092] ⑩Flow cytometry data were analyzed using FlowJo 10.8 software.

[0093] 1.2.4 Plasma metabolomics analysis based on UHPLC / Q-Orbitrap-MS

[0094] 1.2.4.1 Preparation of internal quality control standards

[0095] Select deuterated cholic acid-[d 4 ] as the internal standard (IS). Accurately weigh 1 mg of deuterated cholic acid-[d 4 ], placed in a 1.5 mL centrifuge tube, added 1 mL of 50% methanol aqueous solution to make a 1 mg / mL IS stock solution, and stored at -80°C after aliquoting. Before injection analysis, dilute the IS stock solution with 50% methanol aqueous solution to prepare 5 μg / mL and 500 ng / mL IS working solutions.

[0096] 1.2.4.2 Sample pretreatment

[0097] Take 100 μL of plasma, add 10 μL of IS working solution (5 μg / mL) and 400 μL of glacial acetonitrile, vortex for 5 min, incubate at -20°C for 1 h, centrifuge at 14000 rpm for 10 min at 4°C, transfer all the supernatant to a clean centrifuge tube, blow dry with nitrogen, reconstitute with 100 μL of 50% methanol water, vortex for 5 min, centrifuge at 14000 rpm for 20 min at 4°C, and take the supernatant for UHPLC / Q-Orbitrap-MS metabolomics analysis.

[0098] 1.2.4.3 Instrument conditions

[0099] Metabolite elution was completed on the Ultimate 3000 ultra-high performance liquid chromatography system: the chromatographic column was HSS T3 (Waters, 2.1×100mm, 1.8μm), the mobile phase was 0.1% formic acid in water (A), acetonitrile (B). The elution gradient was as follows: 0-7min, 5%-100% B; 7-10min, 100% B; 10-10.5min, 100%-5% B; 10.5-13min, 5% B. The column temperature was 35°C, the flow rate was 0.3mL / min, and the injection volume was 2μL.

[0100] Mass spectrometry analysis was performed on a Q-ExactiveTM hybrid Q-Orbitrap mass spectrometer system equipped with a heated electrospray ionization source (HESI). The HESI source parameters were set as follows: spray voltage -3.5 kV / +3.5 kV, sheath gas (N 2 )35L / h, auxiliary gas (N 2 )10L / h, purge gas (N 2 )0L / h, capillary temperature 320℃, auxiliary gas heating temperature 350℃.

[0101] Full MS / dd-MS 2 Scanning method, MS 1 The full scan range was m / z 60-900, with a resolution of 70,000; MS 2 The mass spectrometer was scanned over a dynamic mass range with a resolution of 17,500.

[0102] 1.2.4.4 Data Collection

[0103] First, 6 injections of blank samples were collected, and then 6 injections of IS working solution (500 ng / mL) were collected. After the instrument was stable, injection began. The sequence was 1 injection of blank sample interspersed with 5 injections of samples, and samples of different groups were randomly injected.

[0104] 1.2.4.5 Data Analysis

[0105] The raw data obtained by UHPLC / Q-Orbitrap-MS high-resolution mass spectrometry were processed by Xcailbur 4.0 software, and the quasi-molecular ions were controlled within 5 ppm according to the elemental composition provided by the software for metabolite identification. In addition, metabolites were identified by standards, relevant literature, Compound Discoverer 3.2 software (ThermoFisher Scientific), Metlin Metabolite Personal Compound Database, Human Metabolome Database (HMDB, https: / / hmdb.ca / ) and Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov / ).

[0106] Finally, Compound discoverer 3.2 software was used to extract the peak area of ​​small molecule metabolites in plasma. The parameters were set as follows: the scanning mode was positive ion and negative ion; the mass deviation was 5ppm; the maximum drift value of the retention time was 0.2min. After the run was completed, a feature table was generated based on the m / z and retention time of each metabolite, which showed the peak area of ​​each compound in all samples. The data in the table were then normalized by the sum of the peak areas. OPLS-DA (orthogonal partial least squares-discriminant analysis) analysis was performed using SIMCA 14.1 software.

[0107] 1.3 Statistical analysis

[0108] SPSS 26.0 was used to process the baseline data, and the comparison of count data was performed using χ 2 Test, comparison of measurement data, t test was used for those that met the normal distribution, and rank sum test was used for those that did not meet the normal distribution. One-way ANOVA was used for comparison among multiple groups. If the variance homogeneity condition was met, LSD test was selected. If the variance was unequal, Dunnett's T3 test method was selected. Logistic regression analysis was used for correlation analysis. P<0.05 indicated that the difference was statistically significant.

[0109] 2. Results

[0110] 2.1 Proportion of peripheral blood monocyte subsets in ACS patients

[0111] The expression of Mo in peripheral blood of AMI patients at different time points before and after PCI Figure 1 shown.

[0112] AMI patients who undergo PCI have significantly high levels of pro-inflammatory Mo, which prolongs the pro-inflammatory phase and delays the anti-inflammatory repair phase, affecting the repair phase and myocardial repair.

[0113] 2.5.1 Data Quality Control

[0114] The relative standard deviation (RSD) of each group of IS is shown in Table 2. The RSD of all samples was 25.50%, which was less than 30%, indicating that the instrument had good precision during operation, the data set was of high quality, and the results were reliable.

[0115] Table 2 RSD of internal standard

[0116]

[0117] 2.5.2 Multivariate statistical analysis of plasma metabolism changes in peripheral blood monocyte subsets in AMI patients

[0118] The OPLS-DA method with disease supervision model was used for pairwise comparison, and 200 permutation tests were used to verify whether the metabolic model was successfully established.

[0119] like Figure 2 As shown in the figure, there was a significant separation between the Con group and Pre-PCI, Pre-PCI and Post-PCI 1d, Post-PCI 1d and Post-PCI 3d, and Post-PCI 3d and Post-PCI 7d, indicating that at different time points within 1 week before and after PCI, the proportion of peripheral blood Mo subpopulations in AMI patients changed, and the abundance of metabolites in plasma also changed significantly. Multivariate statistical analysis was performed on the plasma metabolome of AMI patients 7 days after PCI and healthy volunteers. The OPLS-DA results showed that there was still a significant separation between AMI patients 7 days after PCI and the Con group ( Figure 3 ), indicating that AMI patients have not recovered to steady-state metabolic levels when the anti-inflammatory recovery period is delayed 7 days after PCI. 2 The intercept is greater than zero, Q 2 The intercept is less than zero, indicating that the metabolic model is not overfitted and is reliable in predicting metabolic differences.

[0120] 2.5.3 Screening and identification of differential metabolites in patients with AMI after PCI when pro-inflammatory Mo levels are high and anti-inflammatory recovery is delayed

[0121] The metabolite data obtained at 3d and 7d post-PCI in AMI patients were normalized and visualized using the fold difference (FC) and false discovery rate (FDR). Biomarkers reflecting the prolonged pro-inflammatory phase after PCI in AMI patients were screened based on FC ≥ 1.2 or FC ≤ 0.67 and FDR < 0.05. The results are as follows: Figure 4 As shown. Red dots indicate significantly upregulated differential metabolites (FC≥1.2, FDR<0.05), and blue dots indicate significantly downregulated differential metabolites (FC≤0.67, FDR<0.05). Differential metabolites were identified by comparing standards, Compound Discoverer software internal databases (mzCloud, Chemspider, etc.), online databases HMDB, Pubchem, and Metlin databases, and the results are shown in Table 3. A total of 16 differential metabolites were identified, among which 10 differential metabolites such as propionylcarnitine, butyrylcarnitine, and valerylcarnitine were at higher levels when the pro-inflammatory Mo level in the peripheral blood of AMI patients was significantly high, but were significantly decreased at 7d Post-PCI, which represented a delay in the anti-inflammatory repair period; while 6 differential metabolites such as linoleoylcarnitine, N-docosahexaenoic acid GABA, and lysophosphatidylcholine (18:2) were at lower levels when the pro-inflammatory Mo level in the peripheral blood of AMI patients was significantly high, but were significantly increased at 7d Post-PCI, which represented a delay in the anti-inflammatory repair period.

[0122] Table 3 List of differential metabolites in AMI patients after 3 days post-PCI vs. after 7 days post-PCI

[0123]

[0124] Note: “a” indicates differential metabolites compared with the standard, and “b” indicates compounds compared with the database.

[0125] The above 16 differential metabolites are mainly divided into four categories: acylcarnitines, lipids, amino acids, and purines. Among them, acylcarnitines account for the largest proportion, with 5 types, namely propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleoylcarnitine.

[0126] 2.5.4 Analysis of metabolic pathways of differential metabolites in patients with high pro-inflammatory Mo levels and delayed anti-inflammatory repair after PCI

[0127] The above 16 differential metabolites were analyzed for metabolic pathway enrichment to explore the changes in metabolic pathways caused by the prolonged pro-inflammatory phase due to high levels of pro-inflammatory Mo in AMI patients after PCI, which affected the changes in plasma differential metabolites during the repair phase. Figure 5As shown in the results, glucose homeostasis, oxidation of branched-chain fatty acids, transport of organic anions, degradation of dietary lipids, β-oxidation of fatty acids, linoleic acid metabolism, and ABC transporter disorders are pathways that are highly correlated with the metabolic changes when the pro-inflammatory phase is prolonged and the anti-inflammatory repair phase is delayed in AMI patients with high levels of pro-inflammatory Mo after PCI. Further summarizing the metabolic pathways of the above changes, it was found that the changes in metabolic pathways when the pro-inflammatory phase is prolonged and the anti-inflammatory repair phase is delayed in AMI patients with high levels of pro-inflammatory Mo after PCI mainly include glucose homeostasis, β-oxidation of fatty acids, lipid metabolism, and ATP energy metabolism.

[0128] 2.5.5 Changes in plasma propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine, and linoleylcarnitine in AMI patients are associated with high levels of proinflammatory monocytes and prolonged proinflammatory phase

[0129] The dynamic metabolic changes of the above five acylcarnitines in the peripheral blood Mo subpopulation of healthy volunteers and AMI patients at different time points before and after PCI were comprehensively analyzed. Figure 6 As shown in Figure 2, the levels of propionylcarnitine, butyrylcarnitine, valerylcarnitine and hydroxyisovalerylcarnitine in plasma at different time points before and after PCI were consistent with the changes in the pro-inflammatory Mo (see Figure 1 ), while the trend of linoleylcarnitine is opposite to the high level of pro-inflammatory Mo (see Figure 1 ).

[0130] 2.5.6 Validation of metabolic changes of propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine, and linoleylcarnitine in UA patients

[0131] OPLS-DA analysis was performed on the changes in plasma metabolism of Mo subgroups in UA patients at different time points before and after PCI. Figure 7 , Figure 8 As shown in the figure, there was a significant separation between the Con group and Pre-PCI, Pre-PCI and Post-PCI 1d, Post-PCI 1d and Post-PCI 3d, Post-PCI 3d and Post-PCI 7d, and Con group and Post-PCI 7d. The results of 200 permutation tests showed that the R 2 The intercept is greater than zero, Q 2 The intercept is less than zero, indicating that the model is not overfitting and has good predictive ability.

[0132] We further analyzed the metabolic changes of propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleylcarnitine in plasma during the changes of peripheral blood Mo subgroups in healthy volunteers, AMI and UA patients at different time points before and after PCI. Fig. 9As shown in the figure, the metabolic changes of the five acylcarnitines in the plasma of UA patients at different time points before and after PCI were similar to those of AMI patients: the metabolic changes of propionylcarnitine, butyrylcarnitine, valerylcarnitine and hydroxyisovalerylcarnitine at different time points before and after PCI were consistent with the change trend of proinflammatory Mo, while the change trend of linoleylcarnitine was opposite to the high level of proinflammatory Mo. Further comparison of the plasma levels of the five acylcarnitines in AMI and UA patients revealed that compared with AMI patients, the plasma levels of propionylcarnitine, butyrylcarnitine, valerylcarnitine and hydroxyisovalerylcarnitine in UA patients were relatively low, which was consistent with the degree of myocardial damage in AMI and UA patients. As for linoleylcarnitine, before PCI, the plasma levels of UA patients were basically the same as those of healthy patients and higher than those of AMI patients, indicating that the plasma linoleylcarnitine content was relatively high when the degree of myocardial damage was relatively mild.

[0133] Example 2

[0134] In this example, the content and changes of propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleoylcarnitine were studied in the C57BL / 6J mouse model of MI / RI, the relationship between the content of linoleoylcarnitine in plasma and heart tissue was analyzed, and the correlation between the accumulation of linoleoylcarnitine in heart tissue and cardiac function was verified.

[0135] 1.3 Experimental animals

[0136] SPF-grade healthy C57BL / 6J male mice were used as research subjects, weighing 25±5g. The animals were provided by Sibeifu (Beijing) Co., Ltd., animal qualification certificate number: SCXK (Beijing) 2019-0010. The experiment was approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine, animal ethics number: TCM-LAEC2023125. All the above animals were raised in the SPF-grade animal room of the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine, with a room temperature of 20-25℃ and a relative humidity of 40-60%. The feed was provided by the animal center and was sterilized granular feed. Drinking bottles filled with clean tap water were placed for mice to drink freely. The drinking bottles were changed daily, and the light and dark environments were alternated for 12 hours each. Before the experiment, all mice were adaptively fed, had free drinking water, had no adverse reactions, and were included in the experiment with normal eating, drinking and activities.

[0137] 1.4 Animal model preparation

[0138] LAD ligation for 45 minutes was selected to prepare the myocardial injury model. The specific steps are as follows:

[0139] 1.4.1 The day before modeling, the mice were skinned and depilated on the neck and chest. After intraperitoneal injection of tribromoethanol anesthesia, the mice were fixed on a heated operating table, intubated and assisted with breathing by a ventilator, with a respiratory rate of 120 times / min and a tidal volume of 0.2 mL.

[0140] 1.4.2 Thoracotomy: After the mouse breathes evenly, make a transverse incision on the chest, use curved forceps to bluntly separate the cortex and muscle tissues, and expose the ribs and intercostal spaces. Clamp the lower edge of the fourth rib with bone forceps, use curved forceps to tilt 20 degrees along the bone forceps into the chest cavity, expand the curved forceps, place a chest expander, and adjust the field of view.

[0141] 1.4.3 Ligation: Before ligation, place a reperfusion thread above the LAD, use a 7-0 suture needle with thread to tie a slipknot 2-3mm below the lower edge of the left atrial appendage to ligate the LAD. The whitening of the myocardium in the left ventricular area indicates that the ligation is successful. Time for 45 minutes, close the chest and exhaust. Use a 5-0 suture needle with thread to suture the skin, disinfect with iodine, and try to stop the ventilator. After the mouse breathes evenly, remove the ventilator and place the mouse on an electric blanket.

[0142] 1.4.4 Reperfusion: 45 minutes after successful ligation, the mouse's cardiac function was tested by ultrasound, and then the slipknot was opened for reperfusion. After reperfusion, the mouse was placed on an electric blanket and waited for awakening.

[0143] 1.5 Experimental Grouping

[0144] Before modeling, the mice were divided into sham operation (Sham) group and model group according to their body weight. All operations in the Sham group were the same as those in the model group, but only needle insertion was performed without ligation. In order to match the time point of clinical blood collection in the first part of the study and considering the self-healing ability of mice, this experiment selected 4 time points, namely 1 day, 3 days, 5 days and 7 days after reperfusion, to further observe the content and changes of propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleylcarnitine in the plasma of MI / RI mice.

[0145] After modeling, all model group mice were divided into 6 groups according to LVEF detected by cardiac ultrasound, namely left anterior descending artery ligation no longer perfused group (LADO), 1 day after reperfusion group (Post-I / R 1d), 3 days after reperfusion group (Post-I / R3d), 5 days after reperfusion group (Post-I / R 5d) and 7 days after reperfusion group (Post-I / R 7d), with 12 mice in each group.

[0146] 1.6 Plasma and tissue sample collection

[0147] 1.6.1 Plasma samples

[0148] Coat the centrifuge tube with EDTA anticoagulant in advance. After the mouse is anesthetized, remove the eyeball to collect blood, mix thoroughly by turning it upside down, and allow the blood to coagulate. After standing at room temperature for 30 minutes, centrifuge at 4°C, 3000 rpm for 10 minutes, collect the supernatant, and store it at -80°C after aliquoting.

[0149] 1.6.2 Tissue samples

[0150] After blood collection, the heart was perfused with pre-cooled saline, and the heart, liver, spleen, lung, and kidney were immediately removed, washed with saline, dried with filter paper, and weighed for calculation of organ index. Three organs were fixed in tissue fixative, and the remaining organs were wrapped with tin foil, placed in cryopreservation tubes, marked, and quickly frozen in liquid nitrogen. After sampling, they were transferred to a -80℃ refrigerator for storage in preparation for other index detection.

[0151] 1.7 Experimental methods

[0152] 1.7.1 Echocardiographic examination of mice

[0153] 45 minutes after modeling and before sampling at each time point, each group of mice underwent cardiac ultrasound examination. The specific method was as follows: the mice were anesthetized and fixed in a supine position on a small animal heating operating table, the skin was prepared (hair removal at the neck and chest), ultrasound coupling agent was applied to the left chest, the probe was close to the left chest, and the detection position was the left side of the sternal midline. After the intact heart appeared in the B-mode ultrasound image field, echocardiograms were collected in the M-mode ultrasound image mode, LVEF was measured, the measurement was repeated for 3 cycles, and the average value was recorded.

[0154] 1.7.2 Preparation of plasma and cardiac tissue samples based on UHPLC / Q-Orbitrap-MS

[0155] Preparation of plasma samples: same as in Example 1.

[0156] Preparation of cardiac tissue samples: Weigh 50 mg of cardiac tissue, add 250 μL of deionized water, homogenize the tissue (low temperature), add 500 μL of ice methanol to the homogenate, and finally a system with a methanol-water ratio of 2:1 (v / v), vortex for 5 min, place on ice for 5 min, centrifuge at 14000 rpm at 4°C for 10 min, transfer all supernatants to clean centrifuge tubes, blow dry with nitrogen, re-dissolve with 50 μL of methanol / water (2:1, v / v), vortex for 5 min, centrifuge at 14000 rpm at 4°C for 20 min, and take the supernatant for UHPLC / Q-Orbitrap-MS analysis. Quality control (QC) sample preparation: Take 10 μL of each sample after reconstitution, mix well, centrifuge and take the supernatant for use.

[0157] 1.7.3 UHPLC / Q-Orbitrap-MS analysis conditions

[0158] Same as Example 1.

[0159] 1.7.4 Data Collection

[0160] First, 6 blank samples were collected, followed by 6 QC samples, and random sampling began after the instrument was stable. The sequence was 1 blank sample and 1 QC sample interspersed every 5 samples.

[0161] 1.7.5 Data Analysis

[0162] The experimental data are expressed as mean ± standard deviation. SPSS26.0 was used for statistical analysis. The t test was used for comparison between two groups, and One-way ANOVA was used for comparison between multiple groups. When the variances were equal, the LSD test method was used, and when the variances were unequal, the Dunnett's T3 test method was used. P<0.05 was considered statistically significant.

[0163] 2 Results

[0164] 2.1 Cardiac function of mice at different time points within 1 week before and after reperfusion

[0165] Ultrasound images of mice at different time points within 1 week before and after reperfusion Fig.10 The measurement results show ( Fig.11 ), compared with those in the Sham group, the LVEF and LVFS of the mice in the LADO group were significantly decreased (P<0.01), and the LVID,d and LVID,s were significantly increased (P<0.05), indicating that the cardiac function of the mice in the LADO group was significantly decreased after modeling. Compared with the LADO group, the LVEF and LVFS of mice in the Post-I / R 1d group were slightly increased, and the LVID,d and LVID,s were decreased, but the differences were not statistically significant (P>0.05); compared with Post-I / R 1d, the LVEF and LVFS of mice in the Post-I / R3d group decreased again, while the LVID,d and LVID,s were not significantly different from those at Post-I / R 1d, and did not show a downward trend, indicating that the cardiac function of mice at Post-I / R 3d was slightly lower than that at Post-I / R1d; compared with the Post-I / R 3d group, the LVEF and LVFS of mice in the Post-I / R 5d and Post-I / R 7d groups showed a gradual upward trend, and the LVID,d and LVID,s also gradually decreased, indicating that the cardiac function had begun to recover on the 5th day of reperfusion. The overall fluctuation trend was consistent with the clinical analysis results, and the model was successfully established.

[0166] 2.2 Changes of propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine, and linoleylcarnitine in mouse plasma at different time points within 1 week before and after reperfusion

[0167] Based on UHPLC / Q-Orbitrap-MS technology, targeted qualitative analysis of propionylcarnitine, butyrylcarnitine, valerylcarnitine, hydroxyisovalerylcarnitine and linoleylcarnitine in mouse plasma at different time points within 1 week before and after reperfusion was performed. Fig.12 As shown in the results, consistent with the trend of clinical results, the levels of propionylcarnitine, butyrylcarnitine and valerylcarnitine increased at 45 minutes after LAD ligation and 3 days after I / R, and decreased at 1 day after I / R and 5 days after I / R. Compared with 5 days after I / R, the levels of propionylcarnitine, butyrylcarnitine and valerylcarnitine decreased at 7 days after I / R; hydroxyisovalerylcarnitine showed more volatile changes than clinical results, but the overall trend was similar to the above three short-chain acylcarnitines; the level of linoleylcarnitine also showed changes similar to the clinical research results. It is worth noting that compared with 45 minutes after LAD ligation, the level of linoleylcarnitine in plasma at 1 day after I / R was slightly increased, but at 3 days after I / R, the level of linoleylcarnitine was decreased again, similar to the clinical results, and continued to increase at 5 days after I / R and 7 days after I / R. Comparing the changes in cardiac function and these five potential biomarkers before reperfusion and 1, 3, 5, and 7 days after reperfusion with the clinical results, it can be seen that 3 days after reperfusion is equivalent to the prolonged pro-inflammatory phase of C57BL / 6J mice, which is consistent with the clinical results.

[0168] 2.3 Relationship between linoleylcarnitine accumulation in cardiac tissue and cardiac function in mice

[0169] The linoleylcarnitine in heart tissue was compared with the standard product, and its primary mass spectrum and secondary fragments were shown in Fig.13 and Fig.14 The results showed that linoleylcarnitine was indeed present in the heart tissue of AMI mice at 3 days of reperfusion.

[0170] The levels of linoleylcarnitine in the heart tissue of mice at 3 days post-I / R were compared with those in the Sham group. Fig.15 As shown in Figure A, compared with the Sham group, the content of linoleylcarnitine in the heart tissue of mice at 3 days after Post-I / R was significantly upregulated (P<0.05). Fig.12 ), the results suggest that linoleylcarnitine does accumulate in cardiac tissue while its content in plasma is low. In order to further verify whether the accumulation of linoleylcarnitine in cardiac tissue is related to myocardial injury, this experiment conducted a correlation analysis between the content of linoleylcarnitine in cardiac tissue and LVEF of mice at 3 days post-I / R. Fig.15As shown in Figure B, as the linoleylcarnitine content in the heart tissue increased, the LVEF of mice decreased, and the correlation coefficient between the two was r=0.816, and P<0.01, indicating that the linoleylcarnitine content in the heart tissue of mice was significantly negatively correlated with LVEF at 3 days of reperfusion.

[0171] Example 3

[0172] This embodiment provides a kit for reflecting high levels of pro-inflammatory Mo, comprising reagents or kits for detecting butyrylcarnitine, valerylcarnitine, propionylcarnitine, hydroxyisovalerylcarnitine and linoleoylcarnitine.

[0173] Example 4

[0174] The present embodiment provides a kit reflecting a high level of pro-inflammatory Mo, comprising a reagent or kit for detecting butyrylcarnitine, valerylcarnitine, propionylcarnitine, hydroxyisovalerylcarnitine, and linoleoylcarnitine, and a reagent or kit for detecting at least one of N-acetyl-L-methionine, lysophosphatidylethanolamine (18:2), lysophosphatidylethanolamine (20:2), lysophosphatidylethanolamine (22:6), L-isoleucine, hypoxanthine, N-docosahexaenoic acid GABA, tetrahydroaldosterone-3-glucuronic acid, lysophosphatidylcholine (18:2), glycocholic acid, and creatinine.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A group of markers that can reflect the prolonged pro-inflammatory phase in ACS patients after PCI, characterized by: Including butyrylcarnitine, valerylcarnitine, propionylcarnitine, hydroxyisovalerylcarnitine and linoleylcarnitine.

2. The marker according to claim 1, characterized in that The markers also include at least one of N-acetyl-L-methionine, lysophosphatidylethanolamine (18:2), lysophosphatidylethanolamine (20:2), lysophosphatidylethanolamine (22:6), L-isoleucine, hypoxanthine, N-docosahexaenoic acid GABA, tetrahydroaldosterone-3-glucuronic acid, lysophosphatidylcholine (18:2), glycocholic acid and creatinine.

3. Use of the marker according to claim 1 or 2 in the preparation of a product reflecting the degree of myocardial injury in ACS patients.

4. Use of a reagent for detecting the marker according to claim 1 or 2 in the preparation of a reagent or a kit for reflecting the degree of myocardial ischemic injury in ACS patients.

5. A method for detecting the marker according to claim 1 or 2 in plasma, characterized in that: The specific steps include: S1. Add internal standard working solution and glacial acetonitrile to the plasma to be tested, mix and incubate at -15 to -20°C for 1 to 1.5 hours, centrifuge at 12000 to 15000 rpm at 3 to 5°C for 20 to 30 minutes, take the supernatant, dry and reconstitute with methanol-water solution, centrifuge at 12000 to 15000 rpm at 3 to 5°C for 20 to 25 minutes, and remove the supernatant as the test solution; the internal standard is deuterated cholic acid-[d4]; S2. Detecting the test solution by liquid chromatography-mass spectrometry technology, and determining the degree of myocardial damage according to the change in the content of the marker.

6. The method according to claim 4, characterized in that The liquid chromatography-mass spectrometry technique adopts UHPLC / Q-Orbitrap-MS.

7. The method according to claim 7, characterized in that: The chromatographic conditions of the UHPLC / Q-Orbitrap-MS include: the chromatographic column is HSS T3, the specifications are 2.1×100 mm, 1.8 μm, the mobile phase A is 0.1% v / v formic acid aqueous solution, the mobile phase B is acetonitrile, and the elution gradient is: 0-7 min, 5%-100% B; 7-10 min, 100% B; 10-10.5 min, 100%-5% B; 10.5-13 min, 5% B; the column temperature is 35° C., and the flow rate is 0.3 mL / min.

8. The method according to claim 7, characterized in that The mass spectrometry analysis of the UHPLC / Q-Orbitrap-MS was performed using a Q-Exactive™ hybrid Q-Orbitrap mass spectrometer system.

9. The method according to claim 8, characterized in that The HESI source parameters for mass spectrometry analysis were: spray voltage -3.5 kV / +3.5 kV, sheath gas 35 L / h, auxiliary gas 10 L / h, sweep gas 0 L / h, capillary temperature 320 °C, and auxiliary gas heating temperature 350 °C.

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